All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Leukemia is a cancer of the blood or bone marrow characterized by an abnormal increase of blood cells, usually leukocytes. Leukemia is clinically and pathologically subdivided into a variety of large groups. The first division is between its acute and chronic forms:
Acute leukemia is characterized by the rapid increase of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Immediate treatment is required in acute leukemia due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. Acute forms of leukemia are the most common forms of leukemia in children.
Chronic leukemia is distinguished by the excessive buildup of relatively mature, but still abnormal, white blood cells. Typically taking months or years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group.
In 2000, approximately 256,000 children and adults around the world developed some form of leukemia, and 209,000 died as a result of it. About 90% of all leukemias are diagnosed in adults.
Pediatric leukemia is the most common type of malignancy in children. Among the leukemia subtypes, acute lymphoblastic leukemia (ALL) is the most frequent [Ries L A G et al., National Cancer Institute, SEER Program. NIH Pub. No. 99-4649. Bethesda, Md., 1999]. During the last 40 years accumulating data regarding the biology of pediatric ALL has improved the ability to assess the situation and adapt the most suitable treatment based on risk classification [Moghrabi A et al., Blood. (2007) 109: 896-904; Schultz K R et al., Blood. (2007); 109: 926-935; Möricke A et al., Leukemia. (2010); 24: 265-284; Gaynon P S et al., Leukemia. (2010); 24: 285-297; Stark B et al., Leukemia. (2010); 24: 419-424].
The Berlin-Frankfurt-Münster (BFM) and the Children Cancer Group (CCG) are examples of such classifications in which patients are divided into risk groups based on their white blood cells count (WBC), response to prednisone at day 8 (d8), age, leukemia cell type (B-lineage or T-cell) and the involvement of adverse chromosomal aberrations such as MLL-rearrangement t(4;11) or Philadelphia chromosome t(9;22) [Schrappe M, Ann Hematol. (2004); 83: S 121-3; Vrooman L M et al., Curr Opin Pediatr. (2009); 21(1):1-8]. Minimal residual disease (MRD) is an indication of the amount of remaining leukemic blasts in a patient's bone marrow (BM) during and/or after treatment, which can be measured by means of flow cytometry (FACS) and polymerase-chain reaction (PCR) [van Dongen J J M et al., Lancet. (1998); 352(9142):1731-1738; Bartram C R, Clin. Chim. Acta. (1993); 217: 75-83]. Currently, the BFM MRD-based protocol is regarded as the most accurate prediction of the chances for disease relapse [Möricke A et al., Leukemia. (2010); 24: 265-284]. Although current knowledge has improved the cure rate up to 80-90%, certain children are still over- or under-treated, resulting in treatment failure [Schrappe M et al., Leukemia. (2010); 24: 253-254; Pui C H and Evans W E, N. Engl. J. Med. (2006); 354: 166-178]. This illustrates the necessity of more knowledge regarding new aspects of ALL biology that will predict disease relapse more accurately. Genetic changes (e.g. Philadelphia chromosome) are already acknowledged as important contributors to ALL development [Smith M et al., J. Clin. Oncol. (1996); 14(1):18-24], however, little is known about the significance of epigenetic changes in pediatric ALL.
The findings presented here provide a link between ALL relapse rates and specific microRNAs (miRNAs or miRs) expression values. More specifically, the present invention demonstrates a link between expression values of miR-151-5p and miR-451, and relapse rates for pediatric ALL and B-ALL.
miRNAs are small (18-24 bp) non-coding RNA molecules that bind the 3′ untranslated region (3′ UTR) of mRNAs to prevent their translation [Bartel D P, Cell. (2004); 116: 281-297]. miRNAs function by either complete complementation with the 3′ UTR that leads to mRNA degradation (similar to siRNA) or by incomplete complementation that results in translational inhibition [Meister G, Cell. (2007); 131: 25-28; He L and Hannon G J, Nat. Rev. Genet. (2004); 5: 522-531; Chen K and Rajewsky N, Nat. Rev. Genet. (2007); 8: 93-103]. The end result of both mechanisms is a decrease in specific protein level, which is thought to be a fine-tuning mechanism of protein expression. For this reason, miRNAs can function as pro-oncogenes or tumor suppressors by preventing the translation of tumor suppressors or oncogenes, respectively [Voorhoeve P M and Agami R, Biochim. Biophys. Acta. (2007). 1775(2):274-82; Chen C Z, N. Engl. J. Med. (2005); 353: 1768-1771; Esquela-Kerscher A and Slack F J, Nature Rev. (2006); 6: 259-269].
In other types of malignant diseases the role of miRNAs was already investigated and certain miRNAs have already been linked to disease prognosis, disease profiling and even became interesting targets for therapy.
An example of such involvement of miRNAs can be demonstrated in normal and malignant hematopoeisis, where miRNA such as miR-150 contributes to normal lymphocytes development by changing its expression levels according to the phase of differentiation to regulate c-MYB translation [Xiao C et al, Cell. (2007); 131:146-59; Kluiver J et al., Leukemia. (2006); 20(11):1931-1936]. On the other hand, miR-150 and miR-96 were reported as downregulated and overexpressed, respectively, in CML [Agirre X et al., Mol Cancer Res (2008); 6(12):1830-1840]. As mentioned before, miRNAs may be used to classify diseases as demonstrated by Mi et al. [Proc. Natl. Acad. Sci. USA. (2007); 104(50):19971-19976] that showed how ALL can be distinguished from AML based on their miRNA profile. Mi et al. demonstrated that miR-451 expression is low in ALL in comparison with AML, thus generally attributing diagnostic, but not prognostic value to miRNA expression patterns in ALL.
Bandres E et al. [Clin. Cancer Res. 2009; 15(7) Apr. 1, 2009] provided a real-time PCR expression analysis of human mature miRNAs on paraffin-embedded tumor samples of gastric cancer stage III. Bandres identified the miRNAs correlated with disease-free and overall survival times, the results were evaluated using other patients, and in vitro cell proliferation and radio-sensitivity studies were performed to support clinical data. Bandres demonstrated that down-regulation of miR-451 was associated with worse prognosis. Over-expression of miR-451 in gastric and colorectal cancer cells reduced cell proliferation and increased sensitivity to radiotherapy. European Patent EP 2,196,543 disclosed methods and kits for prognosis of a predisposition to develop hepatocellular cancer, wherein one of the kits disclosed includes a probe for hsa-miR-451 for said prognosis. Agirre X., et al., [Mol. Cancer Res. 2008, 6(12), December 2008] identified miRNAs potentially implicated in chronic myeloid leukemia (CML). Agirre showed that out of 157 miRNAs tested in mononuclear cells (MNC) and CD34+ cells from 6 patients with CML, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated in CML, whereas hsa-miR-96 was up-regulated in CML cells, compared with cells isolated from healthy donors. Agirre thus showed that low hsa-miR-151 expression is a marker for CML diagnosis when comparing mononuclear cells (MNC) and CD34+ from CML patients with cells derived from healthy patients.
However, stage III gastric cancer in mature, post-operative and post-chemotherapy/radiation therapy patients, hepatocellular cancer patients and CML (myeloid cell line cancer) patients cannot be compared with ALL (lymphoid cell cancer) patients.
Xiuli J. et al., [Ped. Hem. Oncol., 26:1-10, 2009] provided an informative profile of the expression of miRNAs in pre-B-ALL using two independent and quantitative methods: miRNA chip and qRT-PCR of mature miRNA from 40 newly diagnosed pre-B-ALL children. Both approaches showed that miR-222, miR-339, and miR-142-3p were dramatically overexpressed in pre-B-ALL patients, and down regulation of hsa-miR-451 and hsa-miR-373* was confirmed. Thus, Xiuli taught that miR-451 expression is down-regulated in pre-B-ALL patients, in comparison with healthy patients, but only based on a comparison of six healthy and six pre-B-ALL patients, i.e., a small number of patients suffering from B-ALL. Based on this data, Xiuli showed that miR-451 down-regulation is diagnostic of pre-B-ALL, but, as with Mi et al., it was not mentioned or even hinted that miR-451 down regulation is prognostic for B-ALL patients' relapse. Furthermore, the present inventors did not observe a difference in miR-451 expression in B- and T-ALL samples. Specifically, 45% (30/67) and 54% (15/28) of all B-ALL and T-ALL samples, respectively, showed low miR-451 expression, and therefore, according to the present invention it is impossible to discern T-ALL from B-ALL based on miR-451 expression level.
Finally, Fulci V., et al., [Genes, Chromosomes & Cancer 48:1069-1082 (2009)] investigated miRNAs expression profiles in adult ALL patients. miRNA expression was determined by microarray analysis and identified miR-148, miR-151, and miR-424 as discriminative of T-lineage versus B-lineage ALL. While this agrees with some of the inventors' findings, Fulci had found a statistical correlation between miR-151 and B-ALL, but did not make the connection to relapse risk as a function of miR-151 expression.
In pediatric ALL, little is known about the biological role of miRNA and the link between them and ALL was rarely investigated [Schotte D et al., Leukemia. (2009); 23: 313-322]. For this reason, this study focuses on finding miRNA that are involved in pediatric ALL biology with main emphasis on disease relapse. Possible correlation to clinical parameters is sought and potential implementation of those miRNA in disease risk-assessment is evaluated.
The present invention focuses on miRNAs that are involved in pediatric ALL biology, namely miR-451 and miR-151-5p, as well as any combinations thereof, with main emphasis on disease relapse. The inventors demonstrate a correlation between said miRNA expression patterns and clinical parameters allowing the implementation of novel, more accurate risk assessment procedures for ALL relapse. Furthermore, the present invention allows the prognosis to take place at initial diagnosis, rather than later when dependent on diagnostic methods which require lengthy periods. This allows for an early identification of the relapse risk of the patient and the administration of an optimal treatment.
Therefore, one object of the invention is to provide prognostic methods, compositions and kits for the prognosis of ALL.
A more specific object of the invention is to provide prognostic methods, compositions and kits for the prognosis of ALL, monitoring and early detection or diagnosis of ALL relapse, which allow an early accurate prognosis of ALL and provides the necessary information for directing an appropriate treatment regimen from initial diagnosis.
These and other objects of the invention will become apparent as the description proceeds.